Taxifolin

The analysis was previously described by Sokół-Łętowska et al. [51 (link)]. The HPLC-PDA analysis was performed using a Dionex (Germering, Germany) system equipped with the diode array detector model Ultimate 3000, quaternary pump LPG-3400A, autosampler EWPS-3000SI, thermostated column compartment TCC-3000SD, and controlled by Chromeleon v.6.8 software (Thermo Scientific Dionex, Sunnyvale, CA, USA). The Cadenza Imtakt column C5-C18 (75 × 4.6 mm, 5 μm) was used. The mobile phase was composed of solvent C (4.5% aq. formic acid, v/v) and solvent D (100% acetonitrile). The elution system was as follows: 0–1 min 5% D in C, 20 min 25% D in C, 21 min 100% D, 26 min 100% D, 27 min 5% D in C. The flow rate of the mobile phase was 1.0 mL/min and the injection volume was 20 μL. The column was operated at 30 °C. Iridoids were detected at 245 nm, flavan-3-ols at 280 nm, phenolic acids and their derivatives at 320 nm, flavonols, flavanonols, flavones and flavanones at 280 and 360 nm, and anthocyanins at 520 nm.
Loganic acid and its derivatives were expressed as mg of loganic acid equivalents (LAE) per 100 g fresh weight (fw), loganin, sweroside and their derivatives as loganin equivalents (LoE) per 100 g fw, anthocyanins as cyanidin 3-O-glucoside equivalents (CygE) per 100 g fw, derivatives of quercetin and taxifolin as quercetin 3-O-glucoside equivalents (QgE) per 100 g fw, luteolin -O-dihexoside-hexoside as luteolin 7-O-glucoside equivalents (LgE) per 100 g fw, caffeoylquinic acids as mg of 5-O-caffeoylquinic (chlorogenic) acid equivalents (ChAE) per 100 g fw. Solutions of standards (1 mg/ml) were dissolved in 1 mL of methanol. The appropriate amounts of stock solutions were diluted with 50% aqueous methanol (v/v) acidified with 1% HCl in order to obtain standard solutions. Analytical characteristics for determination of phenolic compounds and iridoids are shown in Table S3.

SDS-PAGE and western blot investigations were executed by earlier described protocol with minor amendments [21 (link)]. Subsequent to 24 h and 48 h of administration with TAX at necessary amounts, HCT116 and HT29 cells lyses was carried out in RIPA buffer augmented with freshly added protease and phosphatase inhibitor cocktail 1:100 (Santa Cruz, CA) and concentration of protein was anticipated by Bradford assay [22 ]. To resolve 40-60 μg of protein, 8–12% poly acrylamide gels were used in immune-blotting. Transferred on to a nitrocellulose membrane, with candidate monoclonal primary antibodies, and perceived by super signal west Pico, Dura or Femto Chemiluminescence Reagent (Thermo scientific, USA). Protein bands were quantified by measuring band density using Image J software. The densities of the bands (normalized to actin) relative to that of the untreated control (designated as 1.00) were presented as mean ± SEM of three individual experiments.

Silymarin was obtained from the following suppliers: Sigma-Aldrich (SM 1; St. Louis, MO, USA, batch No. BCBJ0393V), Liaoning Senrong Pharmaceuticals (SM 2; Panjin, China, batch No. 120501), INDENA (SM 3; Settala, Italy, batch No. 32621/M5), Panjin Huacheng Pharmaceutical Company (SM 4; with an additive for better solubility in water, Panjin, China, batch No. E5S66), Takeda (SM 5; Konstanz, Germany, Flavobion^® coated tablets, batch No. 383036), Panjin Huacheng Pharmaceutical Company (SM 6; supplied in August 2019 without batch No., Panjin, China). Flavobion^® coated tablets (25 tablets, 11.360 g) were powdered and subjected to Soxhlet extraction with acetone (450 mL) for 2 h, the extract was evaporated in vacuo to yield the sample SM 5 (126 mg of dry extract). The extract was stored at −80 °C. Standards of silybin A, silybin B, 2,3-cis-silybin A, 2,3-cis-silybin B, 10,11-cis-silybin A, 10,11-cis-silybin A, silychristin A, silychristin B, isosilychristin, silydianin, isosilybin A, isosilybin B, silyhermin, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B, 2,3-dehydrosilychristin A, 2,3-dehydrosilychristin B, 2,3-dehydroisosilybin, and 2,3-dehydrosilydianin were prepared and fully characterized in the Laboratory of Biotransformation, Institute of Microbiology, Prague, CZ [11 (link),21 (link),22 (link),23 (link),24 (link),25 (link)]. Taxifolin was purchased from Amagro (Prague, Czech Republic) and coniferyl alcohol from Sigma-Aldrich (Merck, Kenilworth, NJ, USA). All standard solutions for calibration curves were prepared in dimethyl sulfoxide in volumetric flasks. The silymarin preparations SM 1–SM 6 were also dissolved in dimethyl sulfoxide and their concentrations were 10.3, 6.5, 13.3, 8.3, 15.5, and 23.1 mg/mL, respectively. All substances dissolved in dimethyl sulfoxide were stable during the measurement; no new peaks appeared in repeated measurements even after several weeks. The concentrations of the flavanonol, flavonolignans, and 2,3-dehydroflavonolignans were calculated using seven-point calibration curves.
Silymarin fraction containing concentrated 2,3-dehydroderivatives of flavonolignans was obtained from silymarin (SM 2 preparation) as described previously [23 (link)] using Sephadex LH-20 glass column XK 50 (100 × 5 cm, bead size 25–100 μm, GE Healthcare Life Sciences, Pittsburgh, PA, USA) equipped with a thermostatic jacket (23 °C). Isocratic elution with methanol, flow rate 3 mL/min, volume of each fraction was 30 mL, UV detection at 254 nm, run-time 28 h [23 (link)]. Briefly, 6 g of “silybin free” silymarin (see Appendix B) was loaded onto the column and eluted with methanol to obtain a fraction enriched in 2,3-dehydroderivatives of flavonolignans (typically 0.8 g).
Acetonitrile, methanol, formic acid, dimethyl sulfoxide (Avantor, Radnor, PA, USA), and deionized water were of LC-MS grade.

Taxifolin, registered under the Chemical Abstracts Service (CAS) number 480-18-2, possesses a molecular formula of C15H12O7 and a molecular weight of 304.25 g/mol. Its structural data were obtained from the PubChem organic small molecule bioactivity database (https://pubchem.ncbi.nlm.nih.gov/). The standard Taxifolin was acquired from MedChemExpress (Monmouth Junction, NJ, USA) and stored at room temperature. The stock solution of Taxifolin was prepared by reconstituting the mother liquor to a concentration of 20mM using dimethyl sulfoxide (DMSO), and it was subsequently stored at -20° C for future use.

The three-dimensional structural pattern map of Taxifolin was submitted to PharmMapper (http://www.lilab-ecust.cn/pharmmaDper/), an online website for structural target analysis and screening of pharmacophore. We selected a library of pharmacophore models for screening. To identify the relevant genes associated with pancreatic cancer, we downloaded the gene data obtained from the GeneCards database (https://www.genecards.org/). The intersection of two results yields the target gene.